Monoclonal antibody and hybridoma producing the same

ABSTRACT

The present invention establishes a simple and a highly sensitive detection method for hEPR and provides a novel detection method for human tumors which express hEPR. In particular, the present invention provides a monoclonal antibody which specifically recognizes human epiregulin (hEPR), hybridoma which produces the monoclonal antibody, and a highly sensitive detection method for hEPR using the monoclonal antibody.

TECHNICAL FIELD

The present invention relates to a monoclonal antibody (MoAb) whichspecifically recognizes human epiregulin (hEPR), a hybridoma whichproduces the MoAb and an immunological measurement method tospecifically detect hEPR in samples using a hEPR-polyclonal antibody(hEPR-PoAb) which recognizes hEPR and the MoAb.

BACKGROUND ART

It has been known that there are certain gene abnormalities which occurwith specificity and high frequency in cancer cells, and if a protein(proteins) which is caused by the gene abnormality is found, then such aprotein may be a target molecule for cancer diagnosis and therapy. Amongthem, a group of molecules which belong to epidermal growth factor (EGF)family and their receptor ErbB family molecules have been the targetmolecules for cancer diagnosis and therapy as they are involved in thegrowth of cancer cells and related to malignancy. (J. Clin. Oncol., 17,2639-2648 (1999); Biochem. Biophys. Acta., 1198, 165-184 (1994);Science, 244, 707-712 (1989); Anticancer Res., 20:91-95 (2000); Front.Biosci., 1;6:D685-707 (2001)).

It has been reported that epiregulin (EPR), a member of EGF family, hasa bi-functional character that it promotes the growth of normal cellsand inhibits the growth of certain cancer cells in vitro, and that theexpression level of EPR (mRNA) in normal cells is extremely low exceptin the placenta and monocytes, but high in certain cancer cells (see,for example, The Journal of Biological Chemistry, 1995, Vol. 270, p.7495-7500; The Biochemical Journal, 1997, Vol. 326, p. 69-75).

Further, in the clinical field, it is indicated that epiregulin may be auseful marker for cancer diagnosis because its expression is high incancer of bladder cancer and the pancreatic cancer, (see, for example,Biochemical and Biophysical Research Communications, 2000, Vol. 14, 273(3), p. 1019-1024; AntiCancer Research, 2000, January-February, 20 1A9:p. 91-95; Cancer Research, 2001, Vol. 61, p. 6227-6233).

Up until now, there have been reports that EPR polypeptide is detectedusing polyclonal antibodies (hEPR-PoAb) which recognize human epiregulin(hEPR) (see, for example, The Journal of Biological Chemistry, 1995,Vol. 270, p. 7495-7500; The Biochemical Journal, 1997, Vol. 326, p.69-75), but they are not the methods with high sensitivity and highthroughput because they require concentration operations, labeling withradioisotopes and the like, and there is no report of detecting EPRpolypeptide in the living body. Thus, the establishment of a simple andhighly sensitive method of detecting hEPR may offer useful means, as acancer diagnosis method, for early detection of cancer.

DISCLOSURE OF THE INVENTION

The amino acid sequence homology between EPR and other EGF family memberproteins is known to be 24 to 50% (see The Journal of BiologicalChemistry, 1995, Vol. 270, p. 7495-7500). Furthermore, the sequencehomology of EPR between the species is very high, for example, EPRs ofhuman and mouse are different in only 6 out of 46 amino acid residues.The detection method for EPR by using the polyclonal antibodiesdescribed above requires complicated procedures, and cannotdifferentiate human and mouse EPR. Also, the sensitivity of theconventional detection methods is at most 1 ng/ml, and it is thereforeimpossible to detect EPR in the living body.

The objective of the present invention is to establish a simple andhighly sensitive detection method for hEPR and to utilize the method ofdetecting human tumors. In particular, the present invention provides asimple method of detecting hEPR at a picogram level that is useful fordetecting tumors, and the like.

The present inventors have earnestly studied to solve the problemsdescribed above and finally obtained two hybridomas (1C3, 3E8) producingmonoclonal antibodies (MoAb) which specifically recognize hEPR. Further,sandwich enzyme-linked immunosorbent assay method (S-ELISA) isestablished by combining the MoAb and hEPR-PolyAb, and a detectionsystem, which is highly sensitive and high throughput, is completed.

Thus, the present invention provides following (1) to (9).

(1) A hybridoma producing a monoclonal antibody which specificallyrecognizes human epiregulin (hEPR).

(2) The hybridoma according to (1), which has an accession number ofFERM BP-08647.

(3) The hybridoma according to (1), which has an accession number ofFERM BP-08648.

(4) A monoclonal antibody produced by the hybridoma according to any oneof (1) to (3).

(5) A monoclonal antibody, which recognizes an epitope that isrecognized by the monoclonal antibody produced by the hybridomaaccording to (2) or (3), and specifically recognizes hEPR.

(6) A method of specifically detecting hEPR in a sample in vitro,characterized in that the monoclonal antibody according to (4) and apolyclonal antibody that recognizes hEPR (hEPR-PoAb) are used.

(7) A method of detecting hEPR-expressing cells in vitro, characterizedin that the monoclonal antibody according to (4) is used.

(8) The method according to (7), wherein the hEPR-expressing cells are ahuman tumor.

(9) A kit for detecting a human tumor comprising the monoclonal antibodyaccording to (4).

The contents of the specification and/or drawings of the Japanese PatentApplication No. 2003-070864, which is a basis of the priority of thepresent application, are hereby incorporated by reference in itsentirety.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the results of the measurement of the titer of theanti-hEPR monoclonal antibody which is produced by the hybridoma of thepresent invention by ELISA method.

FIG. 2 shows the results of the measurement of the titer of thebiotinylated-anti-hEPR polyclonal antibody.

FIG. 3 shows the detection sensitivity of a sandwich ELISA (S-ELISA)system using the stepwise method and the same time method.

FIG. 4 shows the results of the specificity test of the present methodusing the 1C3 monoclonal antibody as the first antibody. In the presentsystem, the monoclonal antibody does not react with EGF family moleculesor mouse EPR, and reacts only with hEPR.

FIG. 5 shows the result of the Western blot analysis of hEPR in humanserum. Positions of molecular weight markers (kDa) are shown in the leftside.

FIG. 6 shows the detection of hEPR in human serum by the S-ELISA.

FIG. 7 shows the detection of hEPR in various cell culture media.

BEST MODE FOR CARRYING OUT THE INVENTION

While the present invention is described in detail below, those skilledin the art can practice the present invention in various embodimentsusing known techniques in the art and the present invention is notlimited to the following embodiments.

The term “hEPR” as used herein means a precursor, mature form orfragment of human epiregulin, unless otherwise stated. The “precursor”means a membrane bound form before cleavage by an enzyme, and the“mature form” means the polypeptide with 46 amino acid residues (SEQ IDNO:1) released from the cell membrane. The fragment contains 20 aminoacid residues or more, or preferably with 30 amino acid residues or moreof the polypeptide and must contain a sequence specific to humanepiregulin. The sequence specific to human epiregulin does not mean tobe limited, but includes, for example, a sequence containing the 2nd,11th, 26 to 29th and 39th amino acids of the amino acid sequence of SEQID NO:1, which are known to be different when human epiregulin and mouseepiregulin are compared. The fact that the amino acids at these sitesare different between human and mouse is described in Patent ApplicationWO94/29340 of the same applicant. The fragments having the amino acidsequence containing these sites take different higher structures whenthe EPR fragments derived from human and mouse are compared. “Humanepiregulin” and “mouse epiregulin” are the polypeptides with the sameamino acid sequence as EPR naturally present in human and mouse,respectively. However, they are not necessarily limited to proteinsextracted from the natural sources, but it is intended that theyinclude, for example, recombinants and synthetic polypeptides.

The term “specifically recognize” as used herein means that hEPR isrecognized (or bound), but other proteins of EGF family or non-human EPRsuch as mouse EPR and the like are substantially not recognized (bound).It means, for example, the precursor, mature protein, and fragmentedpolypeptides of hEPR are recognized (bound), but EGF and TGF-α, mouseEPR and the like are substantially not recognized (not bound). Inparticular, it means that its affinity for binding, compared to thatagainst the other proteins of EGF family and non-human EPR such as mouseEPR, is 100 times or higher, preferably 1000 times or higher, morepreferably 10,000 times or higher.

The term “substantially not recognized (bound)” as used herein meansthat the binding is not confirmed by the detection means generally usedin the art or of the present invention. In particular, it means that theprecursor, mature protein and fragmented polypeptide of hEPR exhibit apositive reaction to the sandwich ELISA (S-ELISA) method, the Westernblot method or the like, but EGF and TGF-α of the EGF family and EPRderived from mammals other than human, such as mouse, do not show thereaction (not detected).

The term “antibody” as used herein means a polyclonal antibody (PoAb) ora monoclonal antibody (MoAb) which specifically binds to a complete fulllength molecule of hEPR or a hEPR fragment, or a partial fragment ofthese antibodies (for example, fragments (Fab or F(ab′)2 or Fab′)obtained by digestion with papain or pepsin, or the like) and may beproduced by the method of production described below.

Hybridomas and monoclonal antibodies of the present invention may beproduced as follows. Further, those skilled in the art may use suitablyaltered methods based on the following description and known techniquesin the art.

(1) Preparation of Antigen

The amino acid sequence of hEPR antigen and the nucleotide sequencewhich codes for the same are described in WO94/29340. The amino acidsequence and the nucleotide sequence are shown in SEQ ID NO:1 and SEQ IDNO:2, respectively. Further, EPR is described as a tumor cell growthinhibitory factor in WO94/29340.

Recombinant human EPR may be obtained, as described in WO94/29340, byconstructing an expression vector which contains a fragment of DNA (SEQID NO:2) coding for hEPR and by introducing and expressing the vector ina host cells, for example, not limited but preferably, Bacillus brevis.After the expression using Bacillus brevis, the culture medium isconcentrated by 20 folds using an ultra-filtration membrane (1000 kDa),the pH is adjusted to 7.4 with 1 M Tris-HCl (pH 8.0) and thepurification is carried out using an anion exchange column, for example,a Q-sepharose column. In this case, the pass-through fraction isadjusted to pH 5.0 and further purified by a cation exchange column, forexample, an S-seharose column. Next, purification by a reverse phasecolumn chromatography (C4 column) is carried out so as to obtain asingle band in the electrophoresis.

hEPR may also be synthesized chemically by the solid phase method(Merrifield, J. Am. Chem. Soc., Vol. 85, p 2185 (1963)) based on theamino acid sequence shown in SEQ ID NO:1. Chemical synthesis by thesolid phase method is usually carried out by the standard method usingan automatic peptide synthesizer.

(2) Preparation of Polyclonal Antibodies

A warm-blooded animal is immunized total 4 to 6 times with the antigenpeptide solution alone or together with carriers and diluents. Examplesof the warm-blooded animals used include, for example, rabbits, dogs,guinea pigs, mice, rats and the like. It is preferable to measure theantibody titer by collecting blood specimens after the thirdsubcutaneous immunization. The measurement of antibody titer in theserum is carried out by immobilizing the peptide used as an antigen ontoa 96 well microtiter plate and performing the ELISA method. Afterconfirming that the antibody titer is raised high enough, total blood iscollected, and the antibody is separated and purified by the standardmethod. The purification methods include, for example, ammonium sulfateprecipitation, ion exchange chromatography using an anion exchanger suchas DEAE cellulose and the like, gel-filtration, affinity chromatographywith an active absorbent such as protein A/G and the like, and otherpurification methods. Further, the specificity to hEPR may be increasedby purifying using a column in which hEPR is immobilized on the solidphase.

(3) Preparation of Monoclonal Antibodies

Monoclonal-antibody-producing cells are prepared by selecting fromimmunized warm-blooded animals an individual animal, in which theantibody titer is raised, collecting the spleen or the lymph node 2 to 5days after the last immunization, fusing the antibody-producing cellspresent in these organs with myeloma cells and selecting MoAb producinghybridomas. The fusion is carried out according to the known method suchas the method of Kohler et al. (Nature, 256, 495 (1975)). The myelomacells include but not limited, for example, PAI, P3U1 (Health ScienceResearch Resources Bank; HSRRB), Japan, Catalogue No. JCRB0113 andJCRB0708) and the like. The fusion promotion agents include polyethyleneglycol (PEG) and Sendai virus (HVJ), but preferably PEG with a molecularweight 1000 to 6000. An efficient cell fusion may be achieved by addingthe promotion agent at a concentration of about 10 to 80% and incubatingat 20 to 40° C.

Selection of monoclonal antibody may be carried out in accordance withthe known method. Generally, it is carried out in a cell culture mediumfor animal cells added with HAT (hypoxanthine, aminopterin, thymidine).The media for selection and growth may include, for example, PRMI1640medium containing 10 to 20% bovine fetal serum and the like. Cells aregenerally cultured under 5% CO₂ gas at a temperature 20 to 40° C. for 5days to 3 weeks.

The culture supernatants are collected from wells in which hybridomacells are cultured, and antibodies reacting with the antigen peptide maybe selected by the ELISA method. First, the antigen peptide isimmobilized onto 96-well plates and then blocked with calf serum. Afterreacting the supernatant of hybridomas with Mouse Immunoglobulins/HRP(Amersham-Pharmacia) at 37° C. for 1 hour, color is developed usingTetra Methyl Benzidine Microwell Peroxidase Substrate (TMB; Funakoshi)as a substrate. After terminating the reaction by acid, absorbance at450/540 nm is measured. The antibodies with the absorbance of about 3are selected, and cloning is carried out by the limited dilution method.

The target hybridoma cells thus obtained are cultured and the monoclonalantibody may be obtained from the culture medium. Alternatively, thehybridoma cells may be inoculated intraperitonealy into, for example,mouse (Balb/c) and the monoclonal antibody may be obtained from theascites.

Purification of the monoclonal antibody may be carried out in a similarmanner as the conventional separation and purification of PoAb describedabove.

Further, the present inventors deposited two kinds of hybridomas on Sep.25, 2002 to the International Patent Organism Depositary, NationalInstitute of Advanced Industrial Science and Technology (AIST) (ChuohNo. 6, 1-1-1 Higashi, Tsukuba City, Ibaragi-Ken), with accession Nos.FERM P-19033 and FERM P-19034. Still further, they are moved on Feb. 27,2004 to international depositary based on Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure with accession Nos. FERM BP-08647 and FERMBP-08648.

Furthermore, monoclonal antibodies of the present invention includemonoclonal antibodies which specifically recognize an epitope, which isrecognized by the monoclonal antibodies produced by the hybridomadescribed above, especially the hybridoma with the accession Nos.BP-08647 and FERM BP-08648, and also recognize hEPR. The epitope, whichis recognized by these monoclonal antibodies, contains the amino acidresidues which are included in the unique sequence of hEPR describedabove.

The present invention provides a method of specifically detecting hEPRin a sample in vitro, characterized in that the monoclonal antibodiesand the polyconal antibodies, which recognize hEPR (hEPR-PoAb) of thepresent invention described above, are used. The sample includes blood,body fluids, tissue extracts and the like, collected from subjects.Although not limited, the preferred method is the S-ELISA assay method,comprising a step of contacting a sample, which may contain hEPRantigen, to the monoclonal antibody of the present invention and a stepof contacting the antigen-antibody complex, which is generated in thefirst step, to polyclonal antibodies. The steps described above may becarried out sequentially (stepwise method) or simultaneously (same timemethod). The S-ELISA method is described, for example, in “monoclonalantibody, hybridoma and ELISA” (Iwasaki, Tatsuo et al. KodanshaScientific), and those skilled in the art can practice the method of thepresent invention based on the present description. Since the detectionmethod of the present invention is very sensitive and able to detecthEPR at 10 pg/ml or above, the concentration of hEPR expressed in theliving body (about 20 to 30 pg/ml) is high enough to be detected.

The present invention also provides a method of detecting in vitrohEPR-expressing cells, especially human tumors, characterized in thatthe monoclonal antibodies of the present invention described above areused. Since hEPR is secreted from the cell membrane after beingexpressed in the cells, hEPR may be detected in the extra cellular fluidand there is no need for the sample to contain cells.

The method described above includes a step of contacting a samplederived from human and the monoclonal antibody of the present inventionand a step of detecting the presence of hEPR by determining whether ornot the monoclonal antibody binds to hEPR as an indicator. The targethuman tumors include, for example, cancer of the lung, large intestine,bladder, uterus, colon and the like, but not limited to those.

Since the detection method of the present invention is characterized inthe high sensitivity, the detection can be carried out easily and fastwithout needs for sample manipulations such as concentration and thelike. In the present method, samples containing hEPR at theconcentration of 10 pg/ml or above may be detected by the S-ELISA methodand the like, but it is preferable for the sample to contain 25 pg/ml ormore for sure detection.

Using the detection method of the present invention, it is possible todetermine whether or not the specific tumor cells or the like expresshEPR, and if they do, the level of expression. Further, it may bepossible to contribute elucidating the relation between the expressionof hEPR and the mechanism of tumorigenesis. Still further, based on thedata of tumor cells which express hEPR at a high level and of theexpression level in normal cells, the presence of a tumor in theparticular subject can be detected.

The present invention also provides a kit for detecting human tumorincluding the monoclonal antibody of the present invention.

The kit of the present invention may appropriately include, in additionto the monoclonal antibody of the present invention described above,polyclonal antibodies, a buffer, a coloring agent, a labeling reagent, adiluent and the like. Preferably the kit in the present invention is thekit for performing S-ELISA.

As will be shown below, the present invention is explained moreconcretely with embodiments, but as described above, the presentinvention is not limited to these examples.

EXAMPLE 1 Preparation of Hybridomas

One hundred μl of the recombinant hEPR (1 mg/ml) (prepared by the methoddescribed in WO94/29340) in saline was mixed with an equal volume ofFreund's complete adjuvant, emulsified and inoculated to the back of amouse (Balb/c, 6 weeks of age). After 2 weeks, the mouse wasre-immunized with a mixture of the 50 μl of the saline solution ofantigen peptide (hEPR, 1 mg/ml) and Freund's incomplete adjuvant,emulsified by ultrasonic treatment, and after that, additionalimmunizations were carried out every week. On 40 days afterimmunization, the spleen was removed, the lymphocytes were harvested inPRM1640 medium (supplemented with penicillin and streptomycin) andtreated with 0.17 M ammonium chloride to remove red blood cells.Isolated lymphocytes were fused with myeloma cells P3U1 strain derivedfrom a mouse bone marrow tumor by the polyethylene glycol method(PEG4000) to obtain hybridoma cells. The hybridoma cells thus obtainedwere suspended in HAT medium with feeder cells and then distributed to96-well plates and cultured for 15 days.

EXAMPLE 2 Screening for the Monoclonal Antibody

The culture medium supernatants were recovered from wells in which thehybridoma cells obtained in the Example 1 were cultured, and MoAbs whichreact with the antigen peptide by the ELISA method were selected.

At first, 100 μl of 10 μg/ml antigen peptide was added to each well of96-well plates, immobilized to the solid phase after keeping at 4° C.overnight and blocked with 200 μl of 10% calf serum at 37° C. overnight.One hundred μl of the culture medium supernatant of hybridoma cells wasadded to each well, reacted at 37° C. for 2 hours, and then horse radishperoxidase (HRP)-conjugated anti-mouse antibody (Amersham-Pharmacia),which was diluted 1000 folds, was added and reacted at 37° C. for 1hour. The color was developed using Tetra Methyl Benzidine MicrowellPeroxidase Substrate (TMB; Funakoshi) as a substrate.

After terminating the reaction by adding 100 μl of 4N sulfuric acid,absorbance at 450 to 540 nm was measured, and the MoAbs, 1C3 and 3E8,which show the absorbance of about 3, were selected and cloned by thelimited dilution method.

Mouse (Balb/c), injected with 0.5 ml pristine intraperitoneally 7 daysand 3 days before, were inoculated intraperitoneally with selectedMoAb-producing 1C3 or 3E8 hybridoma cells, and ascites was collectedabout 10 days later. The collected ascites was kept at room temperaturefor 30 minutes, at 4° C. overnight and centrifuged at 15,000×rpm for 10minutes, and then the supernatant was recovered.

The titers of selected 2 kinds of MoAb were measured by the ELISA methodand results were shown in FIG. 1. The amount of recombinant hEPR shownon the horizontal axis was immobilized on a microwell plate, 1C3 or 3E8(1 μg/ml) was added, and after the reaction, color was developed usinghorse radish peroxidase (HRP)-conjugated anti-mouse antibody and TMB.The results indicate that both of the MoAbs reacted in aconcentration-dependent manner.

Further, two hybridomas which produce MoAb 1C3 and 3E8, were depositedon Sep. 25, 2002 to the International Patent Organism Depositary,National Institute of Advanced Industrial Science and Technology (AIST)(Center No. 6, 1-1-1 Higashi, Tsukuba City, Ibaragi-Ken), with theaccession Nos. FERM P-19033 and FERM P-19034.

EXAMPLE 3 Preparation of Polyconal Antibodies

One ml of recombinant hEPR in saline (1 mg/ml) and 1 ml of Freund'scomplete adjuvant (Difco) were mixed and emulsified by ultrasonictreatment, and immunized to the back of a rabbit (Japan white, weight2.7 kg, female; Japan Clea) in 10 separate places or more. One monthlater, 0.5 ml of recombinant hEPR in saline (1 mg/ml) and 0.5 ml ofFreund's incomplete adjuvant (Sigma) were mixed and emulsified byultrasonic treatment, and immunized for the second time in a similarmanner as the first immunization. After the second immunization,additional immunizations were given every week with 1 ml recombinanthEPR in saline (1 mg/ml) and 1 ml of Freund's incomplete adjuvant, whichwere emulsified with ultrasonic treatment. Blood samples were collectedone week after the immunization, kept at room temperature for 1 hourafter stirring with a Pasteur pipette, kept standing at 4° C. overnightand then centrifuged at 5,000×g for 10 minutes to obtain the antiserum.

The antiserum was precipitated with 40% ammonium sulfate, dialyzedagainst 50 mM Tris-HCl (pH 8.0) overnight and purified by a Protein-Gcolumn (Amersham-Pharmacia) to obtain the IgG fraction. To purify theantibodies further, a column was prepared according to the conventionalmethod based on the manufacturer's description by binding 1 mg ofrecombinant hEPR to about 1 ml of NHS-activated Sepharose 4 Fast Flow(Amersham-Pharmacia). The IgG fraction obtained as described above wasadjusted to pH 8.0 and circulated through this column for 10 hours orlonger, and antibodies bound to the column were eluted with 50 mMGlysine-HCl (pH 2.5)/0.15 M NaCl. The eluted antibodies were immediatelybrought to neutral pH with 1 M Tris and dialyzed against 50 mM phosphatebuffer (pH 7.4)/0.15 M to obtain hEPR specific antibodies.

The specific antibodies were biotinylated by a conventional method usingthe biotinylation reagent (Amersham-Pharmacia). The titer ofbiotinylated hEPR-PoAb was measured by the following method. hEPRsolution of the highest concentration 200 ng/ml was diluted by 5 foldand immobilized on a 96 well microplate (100 μl/well). After blockingwith 25% Block Ace (Dainippon Pharmaceutical Co.; 200 μl/well),biotinylated anti hEPR-PoAb (0.125 μg/ml to 1 μg/ml (diluted with 50%Block Ace (Dainippon Pharmaceutical Co.): 100 μl/well) was added andreacted at room temperature for 2 hours, and then Avidin-HRP(Amersham-Pharmacia) diluted 1000 fold was added at 100 μl/well andreacted at room temperature for 30 minutes. After addition of TMB, thereaction was carried out at room temperature for 30 minutes andterminated with 4N sulfuric acid. Absorbance was measured at 450 to 540nm and the titer against the antigen immobilized on the solid phase wasobtained as in Example 2. Results indicate that all of them demonstrateantigen-concentration dependent reactions, confirming that they haveusable titers (FIG. 2).

EXAMPLE 4 Sandwich ELISA System

An ELISA system was constructed using the monoclonal antibody obtainedin Example 2 and the polyclonal antibodies obtained in Example 3.

The monoclonal antibody, 1C3, at 1 μg/ml, was added to a microplate at100 μl/well and incubated at 4° C. for 24 hours to immobilize to thesolid phase. The wells were washed 3 times with 200 μl/well of 50 mMTris-HCl, pH 7.5 containing 0.1% Tween 20 (TBST). Blocking was carriedout by adding 25% Block Ace at 200 μl/well and incubating 4° C. for 24hours.

In the stepwise method, the wells were washed 3 times with TBST 200μl/well, hEPR, serially diluted by 2 fold starting from 1 ng/ml, wasadded and incubated at room temperature for 2 hours, then thebiotinylated anti-hEPR-PoAb (1 μg/ml; 100 μl/well) was added andincubated at room temperature for 2 hours.

In the same time method, the antigen hEPR and biotinylatedanti-hEPR-PoAb were added simultaneously and reacted at room temperaturefor 2 hours.

After washing wells 5 times with TBST, avidine-HPR (Amersham-Pharmacia)diluted 1000 fold was added at 100 μl/well and incubated at roomtemperature for 30 minutes. The reaction was terminated by adding 4Nsulfuric acid and absorbance at 450/540 nm was measured. Resultsindicate that there was no difference in detection sensitivity betweenthe stepwise method and the same time method, and both methods hadsufficient sensitivity (25 pg/ml) for measuring the amount in the livingbody (FIG. 3). In a similar test carried out using 3E8 as the primaryantibody, the results were the same.

EXAMPLE 5 Specificity of the Sandwich ELISA System

The specificity of the present system was confirmed by the method shownin Example 4 (Same time method).

1C3 MoAb (1 μg/ml, 100 μl/well) was immobilized on wells of a microwellplate as the primary antibody, and after blocking with 25% Block Ace(200 μl/well), were added a group of molecules of EGF family member, towhich EPR belongs, (6 factors; epidermal growth factor (EGF),transforming growth factor-α (TGF-α), heparin-binding EGF-like growthfactor (HB-EGF), betacellulin (BTC), amphiregulin (AR), heregulin-α(HRG-α): all purchased from R&D Co. or Sigma Co.), mouse EPR (preparedin the similar way as human EPR), or human EPR, at the amount shown onthe horizontal axis, together with the biotinylated hEPR-PoAb (2 μg/ml,50 μl/well; final 1 μg/ml). The 6 members of EGF family and mouse EPRwere diluted serially in 4 steps, each step by 5 fold, starting from aconcentration of 200 ng/ml and added at 100 μl/well. Similarly, humanEPR was diluted serially in 4 steps, each step by 5 fold, starting froma concentration of 200 pg/ml. After reacting with avidin-HPR, color wasdeveloped using TMB.

As shown in FIG. 4, hEPR was detected with high sensitivity, but othermolecules that were EGF family molecules and mouse EPR were notdetectable even at 1000 times higher concentrations, confirming thespecificity of the present sandwich ELISA system. Similar results wereobtained in the similar test using 3E8 as the primary antibody.

EXAMPLE 6 Western Blot Analysis of Human EPR in Human Serum

Human serum (Rockland INC.) was diluted by 10 fold using 50 mM Tris-HClbuffer pH 7.4 (Gibco BRL). Recombinant hEPR was added to the dilutedhuman serum to prepare test samples by 5 fold serial dilution so thatthe final concentration were in lane 1: 0 pg/ml, lane 2: 8 pg/ml, lane3: 40 pg/ml, lane 4: 200 pg/ml, lane 5: 1000 pg/ml. The test samplesthus obtained were subjected to SDS polyacrylamide gel electrophoresis(SDS-PAGE), transferred to PVDF membrane (Daiichi Pure Chemical Co.),and then analyzed by the immuno-blotting method (J. Biol. Chem., 270,7459-7500) using antibodies, such as anti-hEPR-PoAb, MoAb1C3 and 3E8 (1μg/ml) which recognize hERP. Results indicate that each antibodyspecifically detects hERP in the region of 3.7 to 8.2 kDa (FIG. 5).Although a band of about 28 kDa was detected by PoAb and 1C3 MoAb, itwas regarded to be a non-specific band because the same band wasdetected in the lane of antigen (−).

EXAMPLE 7 Detection of Human EPR in Human Serum by the ELISA Method

The monoclonal antibody, 1C3 or 3E8, at 1 μg/ml was added to the wellsof a microplate by 100 μl/well, and immobilized to solid phase byincubating 4° C. for 24 hours. The plate was washed 3 times with 200μl/well of 50 mM Tris-HCl, pH 7.5 containing 0.1% Tween 20 (TBST) andblocked by adding 200 μl/well of Block Ace and incubating at 4° C. for24 hours. After washing wells 3 times with 200 μl/well of TBST, hEPR in2 fold diluted human serum was serially diluted by 2 fold starting from400 pg/ml and added at 50 μl/well. At the same time the biotinylatedanti-hEPR-PoAb (2 μg/ml) was added at 50 μl/well, and incubated at roomtemperature for 2 hours. After washing 5 times with TBST, 1000 folddiluted Avidin-HRP (Amersham-Pharmacia) was added at 100 μl/well, andreacted at room temperature for 30 minutes. The reaction was terminatedwith 4N sulfuric acid, color was developed with TMB and the absorbancewas measured at 450/540 nm. Results were shown in FIG. 6 indicating thathEPR at a concentration of 25 pg/ml can be detected sufficiently by anyof 1C3 and 3E8.

EXAMPLE 8 Detection of EPR Produced by Cultured Human Cancer Cells

Various culture cells derived from mouse (IC38, RAW264.7, NIH3T3 cloneT7, MoAb3) and human (T-24, A-549, HCT116, colo205, colo201, HeLa, NB69,A431, SK-BR-3, MDA-MB-468, KB, TR-13), were cultured for 3 days in aculture medium containing 10% bovine calf serum starting from 10⁶cells/10 ml/dish, and the culture media were subjected to hEPR detectionby the S-ELISA method.

1C3 MoAb (1 μg/μl) was immobilized to the solid phase at 100 μl/well,and, after blocking, 50 μl of various cell culture media and 50 μl ofthe biotinylated hEPR-PoAb (2 μg/ml) were added to a well simultaneouslyand incubated at room temperature for 2 hours. Hereinafter theoperations were similar to those in Example 4. As controls, the similaroperations were carried out on sera with or without additional hEPR (10%FBS, human serum, 0.1% BSA (hEPR 1 ng/ml), human serum (EPR 1 ng/ml)).

Results indicate that A450 nm was not observed in the culture media frommouse cells such as NIH3T3 clone T7 producing mouse EPR and others,while high production of hEPR was confirmed in human cancer cells suchas A-549 (human lung cancer cells), HCT116 (human colon cancer), colo201(human colon cancer), colo205 (human colon cancer) and T-24 (humanbladder cancer) (FIG. 7), confirming that hEPR in culture media can bespecifically detected. However, hEPR was not detectable in human cancercells such as HeLa (human cervical carcinoma), NB69 (humanneuroblastoma), KB (human epidermal carcinoma (oral cavity)), A431(human epidermal carcinoma (epidermis)), SK-BR-3 (human mammary cancer),MDA-MB-468 (human mammary cancer) and TR-13 (human kidney cancer).

INDUSTRIAL APPLICABILITY

According to the present invention, it becomes possible to provide asystem which detects hEPR in human blood, tissues and the like with highspecificity and sensitivity and is useful for cancer diagnosis and thelike to detect tumor cells expressing hEPR. Further, it is expected thatthe use of the method of the present invention will contribute toelucidation of the presence or absence of the hEPR expression and therelationship between the expression of hEPR and the mechanism oftumorgenesis.

All the publications, patents and patent applications herein referred toare hereby incorporated by references.

1. A hybridoma producing a monoclonal antibody which specificallyrecognizes human epiregulin (hEPR).
 2. The hybridoma according to claim1, which has an accession number of FERM BP-08647.
 3. The hybridomaaccording to claim 1, which has an accession number of FERM BP-08648. 4.A monoclonal antibody produced by the hybridoma according to any one ofclaims 1 to
 3. 5. A monoclonal antibody, which recognizes an epitopethat is recognized by the monoclonal antibody produced by the hybridomaaccording to claim 2 or 3 and specifically recognizes hEPR.
 6. A methodof specifically detecting hEPR in a sample in vitro, characterized inthat the monoclonal antibody according to claim 4 and a polyclonalantibody that recognizes hEPR (hEPR-PoAb) are used.
 7. A method ofdetecting hEPR-expressing cells in vitro, characterized in that themonoclonal antibody according to claim 4 is used.
 8. The methodaccording to claim 7, wherein the hEPR-expressing cells are a humantumor.
 9. A kit for detecting a human tumor comprising the monoclonalantibody according to claim 4.